(a) Cell-cycle Regulatory Proteins
Cell-cycle events are thought to be regulated by a series of interdependent biochemical steps. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. In all eukaryotic cells examined to date, the cell cycle appears to be regulated by the sequential activation of a series of the CDK""s or Cyclin Dependent Kinases (reviewed in Morgan, (1995) Nature 374:131-134; King et al., (1994) Cell 79:563-571; Norbury and Nurse, (1992) Annu. Rev. Biochem. 61:441-470). Yeast cells contain a single CDK known as cdc2 in S. pombe (Beach et al., (1982) Nature 300:706-709; Booher and Beach, (1986) Gene 31:129-134; Hindley and Phear, (1984) Gene 21:129-134; Nurse and Bissett, (1981) Nature 292:558-560; Simanis and Nurse, (1986) Cell 45:261-268; and for review see Forsburg and Nurse, (1991b) Annu. Rev. Cell Riol. 7:227-256) and cdc28 in S. cerevisae. The similarities between the progression of proliferation in mammalian cells and yeast have suggested similar roles for cdc protein kinases across species. In support of this hypothesis, a human cdc2 gene has been found to be able to substitute for the activity of an S. pombe cdc2 gene in both its G1/S and G2/M roles (Lee et al., (1987) Nature 327:31). Likewise, the fact that the cdc2 homolog of S. cerevisae (cdc28) can be replaced by the human cdc2 also emphasizes the extent to which the basic cell-cycle machinery has been conserved in evolution.
The activation of cdc2 kinase activity occurs during the M phase and is controlled at multiple levels involving, among other events, the association with various cyclin subunits and the phosphorylation on threonine 167 by cdc2 activating kinase (CAK) (Booher and Beach, (1987) EMBO J. 6:3441-3447; Booher et al., (1989) Cell 58:485-497; Bueno et al., (1991) Cell 66:149-159; Bueno and Russell, (1993) Mol. Cell Biol. 13:2286-2297; Connolly and Beach, (1994) Mol. Cell Biol. 14:768-776; Fesquet et al., (1993) EMBO J. 12:3111-3121; Forsburg and Nurse, (1991a) Nature 351:245-247; Gould et al., (1991) EMBO J. 3297-3309; Hagan et al., (1988) J. Cell Sci. 91:587-595; Solomon et al., (1992) Mol. Biol. Cell 3:13-27; Solomon et al., (1993) EMBO J. 12:3133-3142). Another well-characterized mechanism of regulating the activity of cdc2 involves its inhibition by phosphorylation of a tyrosine and threonine residues (Tyr-15 and Thr-14) within its ATP binding site (Gould and Nurse, (1989) supra). The inhibitory phosphorylation of cdc2 is mediated at least impart by the weel and mik1 tyrosine kinases (Russel et al., (1987) Cell 49:559-567; Lundgren et al., (1991) Cell 64:1111-1122; Featherstone et al., (1991) Nature 349:808-811; and Parker et al., (1992) PNAS 89:2917-2921). These kinases act as mitotic inhibitors, over-expression of them causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al., (1991) Cell 64:1111-1122).
As the cell is about to reach the end of G2, dephosphorylation of the cdc2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cdc2 complex as a kinase. With the exception of budding yeast and the early embryonic cell divisions of some organisms, the dephosphorylation of tyrosine 15 is a key regulatory step of cdc2 activation (Morla et al., (1989) Cell 58:193-203; Heald et al., (1993) Cell 74:463-474; and for reviews see King et al., (1994) Cell 79:563-571; and Morgan (1995) Nature 374:131-134). A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al., (1991) Cell 67:189-196; Lee et al., (1992) Mol Biol Cell 3:73-84; Millar et al., (1991) EMBO J 10:4301-4309; and Russell et al., (1986) Cell 45:145-153). Cdc25 has been shown to be required for entry into mitosis in a number of different organisms (King et al., 1994). Evidence indicates that both the cdc25 phosphatase and the cdc2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al., (1992) Cell 70:139-151; Smythe et al., (1992) Cell 68:787-797; and Solomon et al., (1990) Cell 63:1013-1024. This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of cdc2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of cdc2 and/or a decrease in the rate of its tyrosine phosphorylation.
In S. pombe, the level of cdc25 oscillates in a cell cycle dependent fashion (Ducommum et al., (1990) Biochem. Biophys. Res. Comm. 167:301-309; Moreno et al., (1990) Nature 344:549-552). Cdc25 accumulates through the cell cycle until mitosis when its level rapidly decreases. The pattern of cdc25 accumulation during the cell cycle is reminiscent of mitotic cyclins which are degraded by the ubiquitin system (Glotzer et al., (1991) Nature 349:132-138; Seufert et al., (1995) Nature 373:78-81).
(b) Ubiquitination Pathways
The ubiquitin-mediated proteolysis system is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin is then transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases.
Many proteins that control cell-cycle progression are short-lived. For example, regulation of oncoproteins and anti-oncoproteins clearly plays an important role in determining steady-state levels of protein expression, and alterations in protein degradation are as likely as changes in transcription and/or translation to cause either the proliferative arrest of cells, or alternatively, the transformation of cells.
The present invention relates to the discovery in eukaryotic cells of novel family of proteins whose apparent function includes a ubiquitin ligase activity. In particular, one feature of members of this family of proteins includes a catalytic domain containing a region homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. The subject proteins are referred to herein collectively as xe2x80x9cpub proteinsxe2x80x9d or xe2x80x9cpub ligasesxe2x80x9d for Protein UBiquitin ligase. As described herein, this family of proteins include at least two paralogous classes of mammalian homologs, xe2x80x9cpub1xe2x80x9d and xe2x80x9cpub2xe2x80x9d. We have cloned at least one human pub1 gene (h-pub1), e.g., a human pub1 protein having an apparent molecular weight of 84.5 kDa, as well as a Schizosaccharomyces pombe pub1 gene (s-pub1) having an apparent molecular weight of 85 kDa. Additionally, we have cloned a human pub2 gene (h-pub2) characterized by an apparent molecular weight of 96.2 kd. The pub proteins have an apparent function in the ubiquitination of, among other cellular proteins, the mitotic activating tyrosine phosphatase cdc25. Accordingly, the subject proteins may be involved in regulating the progression of proliferation in eukaryotic cells by effectively controling the activity of the cdk complexes by modulating the availablity of cdc25.
Moreover, as described in further detail below, the subject pub1 proteins contain a sequence motif (CaLB) which is highly homologous to a consensus sequence which has been implicated in Ca+2-dependent binding to phospholipid membranes in several proteins such as phospholipase A2, PKC and rasGAP.
In S. pombe, disruption of s-pub1 elevates the level of cdc25 protein in vivo increasing the activity of the tyrosine kinases, wee1 and mik1, required to arrest the cell cycle. Loss of wee1 function in an S. pombe cell carrying a disruption in the s-pub1 gene results in a lethal premature entry into mitosis; such lethal phenotype can be rescued by the loss of cdc25 function. An ubiquitin thioester adduct of s-pub1 can be isolated from S. pombe and disruption of s-pub1 dramatically reduces ubiquitination of cdc25. These results indicate that s-pub1 may directly ubiquitinate cdc25 in vivo.
One aspect of the invention features a substantially pure preparation of an h-pub1 polypeptide, e.g., full length or fragments thereof, the full-length form of the h-pub1 protein having an approximate molecular weight in the range of 75-95 kD, preferably about 80-90 kD. In a preferred embodiment: the polypeptide has an amino acid sequence at least 70% homologous to an amino acid sequence represented in SEQ. ID No. 2; the polypeptide has an amino acid sequence at least 80% homologous to an amino acid sequence represented in SEQ. ID No. 2; the polypeptide has an amino acid sequence at least 90% homologous to an amino acid sequence represented in SEQ. ID No. 2; the polypeptide has an amino acid sequence identical to an amino acid sequence represented in SEQ. ID No. 2. In preferred embodiments the fragment comprises at least, for example, 25, 50 or 75 contiguous amino acid residues of SEQ. ID No. 2. For instance, certain embodiments of the subject h-pub1 protein will include a catalytic domain having a ubiquitin ligase activity, and (optionally) all or only a portion of other sequences of the full-length h-pub1, e.g. a calcium-binding domain (CalB motif) and/or an ATP-binding site.
Another aspect of the invention features a substantially pure preparation of an h-pub2 polypeptide, e.g., full length or fragments thereof, the full-length form of the h-pub2 protein having an approximate molecular weight in the range of 85-105 kD, preferably about 90-100 kD. In a preferred embodiment: the polypeptide has an amino acid sequence at least 70% homologous to an amino acid sequence represented in SEQ ID No. 6; the polypeptide has an amino acid sequence at least 80% homologous to an amino acid sequence represented in SEQ ID No. 6; the polypeptide has an amino acid sequence at least 90% homologous to an amino acid sequence represented in SEQ ID No. 6; the polypeptide has an amino acid sequence identical to an amino acid sequence represented in SEQ ID No. 6. In preferred embodiments the fragment comprises at least, for example, 25, 50 or 75 contiguous amino acid residues of SEQ ID No. 6. For instance, certain embodiments of the subject h-pub2 protein will include a catalytic domain having a ubiquitin ligase activity.
Still another aspect of the invention features a substantially pure preparation of an s-pub1 polypeptide, including fragments of the full-length portion, the full-length form of the p85 protein having an approximate molecular weight in the range of 80-90 kD, preferably about 85 kD. In a preferred embodiment: the polypeptide has an amino acid sequence at least 70% homologous to an amino acid sequence represented in SEQ ID No. 4; the polypeptide has an amino acid sequence at least 80% homologous to an amino acid sequence represented in SEQ ID No. 4; the polypeptide has an amino acid sequence at least 90% homologous to an amino acid sequence represented in SEQ ID No. 4; the polypeptide has an amino acid sequence identical to an amino acid sequence represented in SEQ ID No. 4. In preferred embodiments: the fragment comprises at least 25, 50 or 75 contiguous amino acid residues of SEQ ID No. 4. As above, preferred embodiments of the subject s-pub1 protein include a catalytic domain and (optionally) a Calb motif and/or ATP-binding site. However, it will be understood that, for certain uses, only the non-catalytic domains/motifs may be desired.
Polypeptides referred to herein as pub polypeptides, in addition to h-pub1, h-pub2 or s-pub1 further refers to other mammalian paralogs, or other mammalian orthologs.
In general, the biological activity of a pub polypeptide can be characterized as including the ability to transfer an ubiquitin molecule from the relevant ubiquitin conjugating enzyme (UBC) to a residue of a target through a pub ubiquitin thioester intermediate. Moreover, a xe2x80x9cpub biological activityxe2x80x9d also refers to an ability to specifically antagonize the biochemical action of a wild-type pub protein, e.g., a pub protein represented by SEQ ID Nos. 2, 4 or 6. In other words, dominant negative mutants of pub are included within the scope of pub biological activity. Such mutants are exemplified by mutation of the active site cysteine to an alanine or other catalytically inactivating mutant. The biological activity of the pub1 proteins may also include the ability to translocate to specific phospholipid membranes in the presence of calcium and/or to bind a nucleotidyl phosphate such as ATP.
The above notwithstanding, the biological activity of a pub polypeptide may be characterized by one or more of the following attributes: an ability to regulate the cell-cycle of an eukaryotic cell; an ability to modulate proliferation/cell growth of an eukaryotic cell; an ability to modulate entry of a mammalian or yeast cell into M phase; an ability to ubiquitinate a cell-cycle regulator, e.g. a tyrosine phosphatase involved in cell-cycle progression, e.g. a cdc25 phosphatase. Such activities may be manifested by the ability to control the steady state level of cdc25 phosphatase, and thus to control the degree of dephosphorylation of a CDK kinase, e.g. cdc2 or the like. The pub polypeptides of the present invention may also function to modulate differentiation of cells/tissue. The subject polypeptides of this invention may also be capable of modulating cell growth or proliferation by influencing the action of other cellular proteins. A pub polypeptide can be a specific agonist of the function of the wild-type form of the protein, or can be a specific antagonist.
Yet another aspect of the present invention concerns an immunogen comprising a pub polypeptide of the present invention, or a fragment thereof, in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the pub polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response.
Another aspect of the present invention features recombinant h-pub1, h-pub2 or s-pub1 polypeptides, or fragments thereof, having amino acid sequences preferably identical or homologous to the amino acid sequence designated by SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, respectively.
Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an h-pub1 polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID No. 2. In a more preferred embodiment: the nucleic acid encodes a protein having an amino acid sequence at least 80% homologous to SEQ ID No. 2. more preferably at least 90% homologous to SEQ ID No. 2, and most preferably at least 95% homologous to SEQ ID No. 2. The nucleic preferably encodes an h-pub1 protein which specifically transfers an ubiquitin molecule form the relevant UBC to a substrate protein, e.g., cdc25, or specifically antagonizes such ubiquitination.
Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an h-pub1 polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID No. 6. In a more preferred embodiment: the nucleic acid encodes a protein having an amino acid sequence at least 80% homologous to SEQ ID No. 6, more preferably at least 90% homologous to SEQ ID No. 6, and most preferably at least 95% homologous to SEQ ID No. 6. The nucleic preferably encodes an h-pub2 protein which specifically transfers an ubiquitin molecule form the relevant UBC to a substrate protein, e.g., cdc25, or specifically antagonizes such ubiquitination.
Yet another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an s-pub1 polypeptide, or a fragment thereof, having an amino acid sequence at least 70% homologous to SEQ ID No. 4. In a more preferred embodiment: the nucleic acid encodes a protein having an amino acid sequence at least 80% homologous to SEQ ID No. 4, more preferably at least 90% homologous to SEQ ID No. 4, and most preferably at least 95% homologous to SEQ ID No. 4. The nucleic preferably encodes an s-pub1 protein which specifically transfers an ubiquitin molecule form the relevant UBC to a cell cycle regulator, e.g., mitotic activating tyrosine phosphatase, e.g., cdc25.
In another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 25 consecutive nucleotides of SEQ ID Nos. 1, 3 or 6; more preferably to at least 50 consecutive nucleotides of one or both of SEQ ID Nos. 1, 3 or 6; more preferably to at least 75 consecutive nucleotides of SEQ ID No. 1, 3 or 6.
Furthermore, in certain embodiments, the pub nucleic acid will comprise a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the pub gene sequence so as to render the recombinant pub gene sequence suitable for use as an expression vector.
The present invention also features transgenic non-human animals, e.g. mice, which either express a heterologous pub gene, e.g. derived from humans, or which mis-express their own pub gene, e.g. expression is disrupted. Such a transgenic animal can serve as an animal model for studying cellular disorders comprising mutated or mis-expressed pub alleles.
The present invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID Nos. 1, 3 or 6, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Such probes can be used as a part of a diagnostic test kit for identifying transformed cells, such as for measuring a level of a nucleic acid encoding a pub polypeptide in a sample of cells isolated from a patient; e.g. for measuring the mRNA level in a cell or determining whether the genomic pub gene has been mutated or deleted.
Another aspect of the present invention provides a method of determining if a subject, e.g. a human patient, is at risk for a disorder characterized by unwanted cell proliferation, comprising detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a pub gene, e.g., encoding a pub1 protein represented by SEQ ID No. 2, a pub2 protein represented by SEQ ID No. 6, or a homolog thereof; (ii) the mis-expression of the h-pub1 gene. In preferred embodiments: detecting the genetic lesion comprises ascertaining the existence of at least one of a deletion of one or more nucleotides from said gene, an addition of one or more nucleotides to said gene, an substitution of one or more nucleotides of said gene, a gross chromosomal rearrangement of said gene, a gross alteration in the level of a messenger RNA transcript of said gene, the presence of a non-wild type splicing pattern of a messenger RNA transcript of said gene, or a non-wild type level of said protein. For example, detecting the genetic lesion can comprise (i) providing a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of SEQ ID No. 1 or 5, or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the h-pub1 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the h-pub1 gene and, optionally, of the flanking nucleic acid sequences; e.g. wherein detecting the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR); e.g. wherein detecting the lesion comprises utilizing the probe/primer in a ligation chain reaction (LCR). In alternate embodiments, the level of said protein is detected in an immunoassay.
Moreover, the present invention provides a practical approach for the identification of candidate agents able to modulate, e.g., activate or inhibit, ubiquitin-mediated degradation of a cell-cycle regulatory protein in eukaryotic cells, especially yeast and mammalian cells. For instance, the assays permit identification of agents which modulate the ubiquitination of a cell cycle regulatory protein, e.g., a mitotic activating tyrosine phosphatase, e.g., cdc25 phosphatase.
One aspect of the present invention relates to a method for identifying an activator or an inhibitor of ubiquitin-mediated proteolysis of a cell-cycle regulatory protein by (i) providing a ubiquitin-conjugating system that includes the substrate protein, an E3-like complex (e.g., comprising a pub protein a ligase activity thereof), and ubiquitin under conditions which promote the ubiquitination of the target protein, and (ii) measuring the level of ubiquitination of the subject protein brought about by the system in the presence and absence of a candidate agent. For example, a decrease in the level of ubiquitin conjugation is indicative of an inhibitory activity for the candidate agent. The level of ubiquitination of the regulatory protein can be measured by determining the actual concentration of protein:ubiquitin conjugates formed; or inferred by detecting some other quality of the subject protein affected by ubiquitination, including the proteolytic degradation of the protein. In certain embodiments, the present assay comprises an in vivo ubiquitin-conjugating system, such as a cell able to conduct the regulatory protein through at least a portion of a ubiquitin-mediated proteolytic pathway. In other embodiments, the present assay comprises an in vitro ubiquitin-conjugating system comprising a reconstituted protein mixture in which at least the ability to transfer ubiquitin to the regulatory protein is constituted.
Still another approach relies on a competitive binding assay to detect potential modulatory agents. For example, the ability of all or a portion of the pub protein to bind to cdc25 (or another cellular substrate protein) or other components of the ubiquitin pathway (e.g. E2""s) can be assessed in the presence and absence of a test agent. In similar fashion, the ability of a test agent to modulate the function of the CaLB motif of a pub1 protein can be assessed.
The present invention also provides a method for producing a hyper- or a hypo-proliferative cell, e.g., a cell which has an impaired cell-cycle checkpoint such as the premature progression of the cell through at least a portion of a cell-cycle. As an example, a hyper-proliferative cell, e.g., a transformed mammalian cell, can be produced by disrupting a pub gene or gene product. Such cells are useful for identifying agents that modulate proliferation such as mitotic inhibitors, e.g., agents which may inhibit at least one regulatory protein of the cell cycle in a manner which counter-balances the effect of the impairment.
The impaired checkpoint can be generated, for example, by molecular biological, genetic, and/or biochemical means. The checkpoint to be impaired can comprise a regulatory protein or proteins which control progression through the cell-cycle, such as those which control the G2/M transition. By way of example, the impaired checkpoint can comprise a pub protein which controls the ubiquitination of a cdc25 phosphatase, and thus the degree of dephosphorylation of a CDK protein kinase, such as cdc2.
In another embodiment, cells impaired in a mitotic checkpoint can also be created by using agents which disrupt the binding of a pub protein to at least one of its targets, e.g., a cdc25 phosphatase. Such a system can be used to modulate cell proliferation and/or growth. In one embodiment, the method comprises administering a pub mimetic, e.g. a peptidomimetic, which binds to a cdc25 phosphatase, and inhibits the interaction between that protein and a pub ligase.
Furthermore, humanized yeast cells can be generated so as to comprise heterologous cell-cycle proteins (i.e. cross-species expression). For example, an exogenous pub can be expressed in a Schizosaccharomyces cell, such as Schizosaccharomyces pombe carrying a null mutation of the pub gene. The exogenous pub can be, for example, the human pub homolog described herein. Humanized yeast cells can provide useful assays for screening modulators, e.g., activators or inhibitors, of proliferation in vivo.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
The cyclin dependent kinases are subject to multiple levels of control. One well-characterized mechanism regulating the activity of cdks involves the phosphorylation of tyrosine, threonine, and serine residues; the phosphorylation level of which varies during the cell-cycle (Draetta et al. (1988) Nature 336:738-744; Dunphy et al. (1989) Cell 58:181-191; Morla et al. (1989) Cell 58:193-203; Gould et al. (1989) Nature 342:39-45; and Solomon et al. (1990) Cell 63:1013-1024). The phosphorylation of cdc2, for example, on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of cdc2 is mediated at least impart by the wee1 and mik1 tyrosine kinases (Russel et al. (1987) Cell 49:559-567; Lundgren et al. (1991) Cell 64:1111-1122; Featherstone et al. (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64:1111-1122).
Dephosphorylation of the cdk-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cdk/cyclin complex as a kinase. A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189-196; Lee et al. (1992) Mol Biol Cell 3:73-84; Millar et al. (1991) EMBO J 10:4301-4309; and Russell et al. (1986) Cell 45:145-153). Recent evidence indicates that both the cdc25 phosphatase and the cdk-specific tyrosine kinases (wee1/mik1) are detectably active during the cell-cycle, suggesting that there is an ongoing competition between these two activities to fine tune cell-cycle progression (Kumagai et al. (1992) Cell 70:139-151; Smythe et al. (1992) Cell 68:787-797; and Solomon et al. (1990) Cell 63:1013-1024.
The role of the ubiquitin dependent proteolytic pathway in the regulation of cdc25 has been examined by us both in vivo and in vitro. We have observed that cdc25A can be ubiquitinated in vitro, which ubiquitination requires an active E1 enzyme. Furthermore, we have found that the level of cdc25 protein increases upon inactivation of a temperature sensitive E1 gene. In addition, poly-ubiquitinated cdc25 can be detected in cells overexpressing a histidine-tagged ubiquitin gene. Finally, inhibition of the 26S proteosome with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal (LLnL) leads to the accumulation of the phosphorylated form of cdc25. Moreover, results from in vitro ubiquitination reactions support the notion that phosphorylation of cdc25 may be a necessary prerequisite for ubiquitination. This finding is likely to be physiologically relevant to the regulated degradation of cdc25, because it is the phosphorylated form of cdc25 which is active as a protein phosphatase.
The specificity of the ubiquitination reaction is thought to be conferred at least in part by the E3 protein. We therefore sought to clone the E3 ligase(s) which specifically target :cdc25 for ubiquitin-dependent degradation. The present invention makes available nucleic acids encoding gene products which play a role in the ubiquitinylation of cdc25, and perhaps other regulatory proteins. Accordingly, the subject gene products may effect growth of eukaryotic cells by functioning as a tumor suppressor which down regulates mitotic acitivation by cdc25. Given the prominence of the cdc25 regulatory pathways in various aspects of cell growth, and probably differentiation, a salient feature for each of the subject nucleic acids, polypeptides, antibodies, and derivatives thereof, includes both therapeutic and diagnostic uses. Moreover, drug screening assays are described herein which provide a systematic and practical approach for identifying candidate agents able to modulate, e.g., activate or inhibit, ubiquitin-mediated degradation of a cell-cycle regulatory protein, such as the mitotic activating tyrosine phosphatase cdc25, in the eukaryotic cells, e.g. mammalian, e.g., human cells.
In particular, as described in the appended examples, the present invention describes the cloning of novel proteins containing a region homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP and other ubiquitin ligases. The proteins which are the subject of the present invention are referred to herein collectively as xe2x80x9cpubxe2x80x9d proteins for protein ubiquitin ligases. As described herein, these proteins include a yeast pub gene product and several human homologs. For example, we have cloned the genes for a human pub protein, referred to herein as xe2x80x9ch-pub1xe2x80x9d, having an apparent molecular weight of 84.5 kDa (h-pub1), as well as a Schizosaccharomyces pombe homolog, xe2x80x9cs-pub1xe2x80x9d, having an apparent molecular weight of 85 kDa. In addition, we have cloned other pub paralogs from human cDNA libraries, such as the 96.2 kd xe2x80x9ch-pub2xe2x80x9d polypeptide described below. The nucleic acid and amino acid sequences, respectively, for each of the exemplary pub proteins are provided in the appended sequence listing as follows: SEQ ID No. 1 and 2 for h-pub1, SEQ ID No. 3 and 4 for s-pub1, and SEQ ID No. 5 and 6 for h-pub2.
The pub proteins apparently play a role in the ubiquitination of regulatory proteins, such as the mitotic activating tyrosine phosphatase cdc25, and thus they may regulate the progression of proliferation in eukaryotic cells by regulating the activity of cdk complexes. All known protein ubiquitin ligases (E3s) contain a carboxyl terminal xe2x80x9chectxe2x80x9d domain (for homologous to E6-AP carboxyl terminus). See Huibregtse et al. (1995) PNAS 92:2563-2567. The hect domain for s-pub1 corresponds to Tyr662-Glu766 of SEQ ID No. 4, while the hect domain of h-pub1 is provided by Ile639-Glu735 of SEQ ID No. 2, and the hect domain pf h-pub2 is represented in Ile727-Asp834 of SEQ ID No. 6. The active site cysteine resides in the hect domain (Cys734 for s-pub1, Cys703 for h-pub1, and Cys801 for h-pub2).
Both h-pub1 and h-pub2 share about 50 percent homology with the hect domain of s-pub1. The fussion yeast pub1 protein apparently has two additional motifs, an ATP binding motif (Gly84-Gly89) and a calcium lipid binding domain (Leu20-Asn67; termed here a xe2x80x9cCaLBxe2x80x9d motif) which is highly homologous to a consensus sequence implicated in: Ca+2 dependent binding to phospholipid membranes in several proteins such as phospholipase A2, PKC and rasGAP. Both the CaLB and ATP binding domains of s-pub1 are conserved in h-pub1 (see SEQ ID No. 2, Leu19-Ser59 for CaLB motif, and Gly77-Gly82 for ATP binding motif), but not apparently in h-pub2.
In S. pombe, disruption of s-pub1 elevates the level of cdc25 protein in vivo. Loss of wee1 function in an S. pombe cell carrying a disruption in the s-pub1 gene results in a lethal premature entry into mitosis; such lethal phenotype can be rescued by the loss of cdc25 function. An ubiquitin thioester adduct of s-pub1 can be isolated from S. pombe and disruption of s-pub1 dramatically reduces ubiquitination of cdc25. These results suggest that s-pub1 may directly ubiquitinate cdc25 in vivo. Human pub1 was found to complement the loss of the fission yeast gene and restore the cell size at mitosis to wild-type. This indicates that h-pub1 is a biologically active, functional homolog of yeast pub1.
For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
The terms peptides, proteins and polypeptides are used interchangeably herein.
As used herein, the term xe2x80x9cnucleic acidxe2x80x9d refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
As used herein, the term xe2x80x9cgenexe2x80x9d or xe2x80x9crecombinant genexe2x80x9d refers to a nucleic acid comprising an open reading frame encoding a pub polypeptide of the present invention, including both exon and (optionally) intron sequences. A xe2x80x9crecombinant genexe2x80x9d refers to nucleic acid encoding a pub polypeptide and comprising pub-encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal pub gene or from an unrelated chromosomal gene. An exemplary recombinant genes encoding the subject pub poypeptides is represented by any of SEQ ID Nos: 1, 3 or 5. The term xe2x80x9cintronxe2x80x9d refers to a DNA sequence present in a given pub gene which is not translated into protein and is generally found between exons.
As used herein, the term xe2x80x9ctransfectionxe2x80x9d means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. xe2x80x9cTransformationxe2x80x9d, as used herein, refers to a process in which a cell""s genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a pub polypeptide of the present invention or where anti-sense expression occurs from the transferred gene, the expression of a naturally-occurring form of the pub protein is disrupted.
As used herein, the term xe2x80x9cvectorxe2x80x9d refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as xe2x80x9cexpression vectorsxe2x80x9d. In general, expression vectors of utility in recombinant DNA techniques are often in the form of xe2x80x9cplasmidsxe2x80x9d which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, xe2x80x9cplasmidxe2x80x9d and xe2x80x9cvectorxe2x80x9d are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
xe2x80x9cTranscriptional regulatory sequencexe2x80x9d is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant pub gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the pub protein.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of a urogenital origin, e.g. renal cells, or cells of a neural origin, e.g. neuronal cells. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal, preferably a non-human mammal, a bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of a pub protein, e.g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant pub gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. The xe2x80x9cnon-human animalsxe2x80x9d of the invention include vertebrates such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding, for example, embryogenesis and tissue patterning. The term xe2x80x9cchimeric animalxe2x80x9d is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal. The term xe2x80x9ctissue-specific chimeric animalxe2x80x9d indicates that the recombinant pub gene is present and/or expressed in some tissues but not others.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., a pub polypeptide), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
As is well known, genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity. The term xe2x80x9cDNA sequence encoding a pub polypeptidexe2x80x9d may thus refer to one or more genes within a particular individual. Moreover, certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
xe2x80x9cHomologyxe2x80x9d refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
xe2x80x9cCells,xe2x80x9d xe2x80x9chost cellsxe2x80x9d or xe2x80x9crecombinant host cellsxe2x80x9d are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A xe2x80x9cchimeric proteinxe2x80x9d or xe2x80x9cfusion proteinxe2x80x9d is a fusion of a first amino acid sequence encoding the subject pub polypeptide with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the pub polypeptide. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an xe2x80x9cinterspeciesxe2x80x9d, xe2x80x9cintergenicxe2x80x9d, etc. fusion of protein structures expressed by different kinds of organisms.
The term xe2x80x9cevolutionarily related toxe2x80x9d, with respect to nucleic acid sequences encoding pub, refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants. The term also refers to nucleic acid sequences which, while derived from a naturally occurring pub genes, have been altered by mutagenesis, as for example, combinatorial mutagenesis described below, yet still encode polypeptides which have at least one activity of a pub protein.
The term xe2x80x9cisolatedxe2x80x9d as also used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule. For example, isolated nucleic acids encoding the subject pub polypeptides preferably include no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks particular pub gene in genomic DNA, more preferably no more than 5 kb of such naturally occurring flanking sequences, and most preferably less than 1.5 kb of such naturally occurring flanking sequence. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an xe2x80x9cisolated nucleic acidxe2x80x9d is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
As used herein, a xe2x80x9cmitotic activating tyrosine phosphatasexe2x80x9d refers to a phosphatase which is involved in one or more aspects of cell-cycle progression, e.g., progression from G0 to G1, G1 to S phase and/or G2 to M phase.
The term xe2x80x9cE3-like complexxe2x80x9d refers to a protein complex including a pub protein ubiquitin ligase and other associated proteins, which protein complex augments or otherwise facilitates the ubiquitination of a protein. In preferred embodiments, the E3-like complex includes a pub protein which is capable of ubiquitinating the mitotic tyrosine phosphatase cdc25.
As used herein xe2x80x9cE3-likexe2x80x9d or xe2x80x9cpub-dependent ubiquitinationxe2x80x9d refers to the conjugation of ubiquitin to a protein by a mechanism which requires a pub ligase activity.
The term xe2x80x9csubstrate proteinxe2x80x9d or xe2x80x9ctarget proteinxe2x80x9d refers to a protein, preferably a cellular protein, which can be ubiquitinated by a pub-dependent reaction pathway.
The term xe2x80x9cwhole lysatexe2x80x9d refers to a cell lysate which has not been manipulated, e.g. either fractionated, depleted or charged, beyond the step of merely lysing the cell to form the lysate. The term whole cell lysate does not, however, include lysates derived from cells which produce recombinant forms of one or more of the proteins required to constitute a ubiquitin-conjugating system for pub-dependent ubiquitination of a target protein.
The term xe2x80x9ccharged lysatexe2x80x9d refers to cell lysates which have been spiked with exogenous, e.g., purified, semi-purified and/or recombinant, forms of one or more components of a pub-dependent ubiquitin-conjugating system, or the target protein thereof. The lysate can be charged after the whole cells have been harvested and lysed, or alternatively, by virtue of the cell from which the lysate is generated expressing a recombinant form of one or more of the conjugating system components.
The term xe2x80x9csemi-purified cell extractxe2x80x9d or, alternatively, xe2x80x9cfractionated lysatexe2x80x9d, as used herein, refers to a cell lysate which has been treated so as to substantially remove at least one component of the whole cell lysate, or to substantially enrich at least one component of the whole cell lysate. xe2x80x9cSubstantially removexe2x80x9d, as used herein, means to remove at least 10%, more preferably at least 50%, and still more preferably at least 80%, of the component of the whole cell lysate. xe2x80x9cSubstantially enrichxe2x80x9d, as used herein, means to enrich by at least 10%, more preferably by at least 30%, and still more preferably at least about 50%, at least one component of the whole cell lysate compared to another component of the whole cell lysate. The component which is removed or enriched can be a component of a ubiquitin-conjugation pathway, e.g., ubiquitin, a target protein, an E1, an E2, an E3-like complex, a cdc25 phosphatase, and the like, or it can be a component which can interfere with a ubiquitin-binding assay, e.g., a protease.
The term xe2x80x9csemi-purified cell extractxe2x80x9d is also intended to include the lysate from a cell, when the cell has been treated so as to have substantially more, or substantially less, of a given component than a control cell. For example, a cell which has been modified (by, e.g., recombinant DNA techniques) to produce none (or very little) of a component of a ubiquitin-conjugation pathway, will, upon cell lysis, yield a semi-purified cell extract.
The term xe2x80x9ccomponent of a ubiquitin-conjugation pathwayxe2x80x9d, as used herein, refers to a component which can participate in the ubiquitination of a target protein either in vivo or in vitro. Exemplary components of a ubiquitin-conjugation pathway include ubiquitin, an E1, an E2, an E3-like complex, a target protein, and the like.
By xe2x80x9csemi-purifiedxe2x80x9d, with respect to protein preparations, it is meant that the proteins have been previously separated from other cellular or viral proteins. For instance, in contrast to whole cell lysates, the proteins of reconsituted conjugation system, together with the target protein, can be present in the mixture to at least 50% purity relative to all other proteins in the mixture, more preferably are present at at least 75% purity, and even more preferably are present at 90-95% purity.
The term xe2x80x9cpurified proteinxe2x80x9d, with respect to components of the ubiquitination pathway, refers to a preparation of a protein or proteins which are preferably isolated from, or otherwise substantially free of, other proteins normally associated with the protein(s) in a cell or cell lysate. The term xe2x80x9csubstantially free of other cellular proteinsxe2x80x9d (also refered to herein as xe2x80x9ccontaminating proteinsxe2x80x9d) is defined as encompassing individual preparations of each of the component proteins comprising less than 20% (by dry weight) contaminating protein, and preferably comprises less than 5% contaminating protein. Functional forms of each of the component proteins can be prepared as purified preparations by using a cloned gene as described in the attached examples. By xe2x80x9cpurifiedxe2x80x9d, it is meant, when referring to the component proteins preparations used to generate the reconstituted protein mixture, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of the component proteins either as purified preparations or in their function in the subject reconstituted mixture). The term xe2x80x9cpurifiedxe2x80x9d as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term xe2x80x9cpurexe2x80x9d as used herein preferably has the same numerical limits as xe2x80x9cpurifiedxe2x80x9d immediately above. xe2x80x9cIsolatedxe2x80x9d and xe2x80x9cpurifiedxe2x80x9d do not encompass either protein in its native state (e.g. as a part of a cell), or as part of a cell lysate, or that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins) substances or solutions. The term isolated as used herein also refers to a component protein that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
As described below, one aspect of the invention pertains to isolated nucleic acid having a nucleotide sequence encoding a pub protein, and/or equivalents of such nucleic acids. The term nucleic acid as used herein is intended to include fragments and equivalents. The term equivalent is understood to include nucleotide sequences encoding functionally equivalent pub proteins or functionally equivalent polypeptides which, for example, retain the ability to bind to a mitotic activating tyrosine phosphatase. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants; and will, therefore, include sequences that differ from the nucleotide sequence of the gene encoding h-pub1 shown in SEQ ID No: 1 or the gene encoding s-pub1 shown in SEQ ID No: 3 or the h-pub2 sequence shown in SEQ ID No. 5, due to the degeneracy of the genetic code. Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27xc2x0 C. below the melting temperature (Tm) of the DNA duplex formed in about 1M salt) to the nucleotide sequence of pub gene represented in SEQ ID No: 1, SEQ ID No: 3 or SEQ ID No. 5. In one embodiment, equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 3 or SEQ ID No. 5.
Moreover, it will be generally appreciated that, under certain circumstances, it may be advantageous to provide homologs of the subject pub proteins, which homologs function in a limited capacity as one of either an agonists (mimetic) or an antagonist in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein. Thus, specific biological effects can be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all of a pub proteins""s biological activities. For instance, antagonistic homologs can be generated which interfere with the ability of the wild-type (xe2x80x9cauthenticxe2x80x9d) pub1 protein to associate with cdc25 phosphatase, but which do not substantially interfere with the formation of complexes between pub1 and other cellular proteins, such as may be involved in other regulatory mechanisms of the cell.
Polypeptides referred to herein as pub polypeptides preferably have an amino acid sequence corresponding to all or a portion of the pub1 amino acid sequence shown in SEQ ID No. 2 or in SEQ ID No.4, or the pub2 amino acid sequence shown in SEQ ID No. 6, or are homologous with one of these proteins, such as other human paralogs, or mammalian orthologs. In general, the biological activity of a pub polypeptide will be characterized as including the ability to transfer an ubiquitin molecule form the relevant ubiquitin conjugating enzyme (UBC) to a lysine residue of its target through a pub ubiquitin thioester intermediate; and an ability to translocate to specific phospholipid membranes in the presence of calcium. The above notwithstanding, the biological activity of a pub polypeptide may be characterized by one or more of the following attributes: an ability to regulate the cell-cycle of an eukaryotic cell, especially a mammalian cell (e.g., of a human cell), or a yeast cell such as a Schizosaccharomyces cell; an ability to modulate proliferation/cell growth of a eukaryotic cell; an ability to modulate entry of a mammalian or yeast cell into M phase; an ability to ubiquitinate a cell-cycle regulator, e.g. a mitotic activating tyrosine phosphatase, e.g. cdc25. Such activities may be manifested by the ability to control the steady state level of cdc25 phosphatase, and thus to control the degree of dephosphorylation of a cyclin dependent kinase. The pub polypeptides of the present invention may also function to modulate differentiation of cells/tissue. The subject polypeptides of this invention may also be capable of modulating cell growth or proliferation by influencing the action of other cellular proteins. A pub polypeptide can be a specific agonist of the function of the wild-type form of the protein, or can be a specific antagonist, such as a catalytically inactive mutant. Other biological activities of the subject pub proteins are described herein, or will be reasonably apparent to those skilled in the art in light of the present disclosure.
In one embodiment, the nucleic acid of the invention encodes a polypeptide which is an agonist or antagonist of the naturally occurring h-pub1 protein and comprises an amino acid sequence identical or homologous to the amino acid sequence represented in SEQ ID No. 2. Preferred nucleic acids encode a polypeptide at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in SEQ ID No. 2. Nucleic acids which encode polypeptides having an activity of a p19 protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID No. 2 are also within the scope of the invention. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence encoding an h-pub1 protein shown in SEQ ID No. 2. A preferred portion of the cDNA molecule designated by SEQ ID No. 1 includes the coding region of the molecule.
In one embodiment, the nucleic acid of the invention encodes a polypeptide which is an agonist or antagonist of the naturally occurring h-pub2 protein and comprises an amino acid sequence identical or homologous to the amino acid sequence represented in SEQ ID No. 6. Preferred nucleic acids encode a polypeptide at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in SEQ ID No. 6. Nucleic acids which encode polypeptides having an activity of a p19 protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID No. 6 are also within the scope of the invention. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence encoding an h-pub2 protein shown in SEQ ID No. 6. A preferred portion of the cDNA molecule designated by SEQ ID No. 5 includes the coding region of the molecule.
In another embodiment, the nucleic acid of the invention encodes a polypeptide which is an agonist or antagonist of the naturally occurring s-pub1 protein and comprises an amino acid sequence identical or homologous to the amino acid sequence represented in SEQ ID No. 4. Preferred nucleic acids encode a polypeptide at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in SEQ ID No. 4. Nucleic acids which encode polypeptides having an activity of an s-pub1 protein and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID No. 4 are also within the scope of the invention. Preferably, the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence encoding an s-pub1 protein shown in SEQ ID No. 4. A preferred portion of the cDNA molecule shown in SEQ ID No. 3 includes the coding region of the molecule.
Isolated nucleic acids which differ from the nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 3 or SEQ ID No. 5 due to degeneracy in the genetic code are also within the scope of the invention. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in xe2x80x9csilentxe2x80x9d mutations which do not affect the amino acid sequence of the protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject pub proteins will exist among mammalian cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding a particular pub protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
Fragments of the nucleic acid encoding a biologically active portion of the subject pub proteins are also within the scope of the invention. As used herein, a fragment of the nucleic acid encoding an active portion of a pub protein refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the full length amino acid sequence of, for example, the pub protein represented in SEQ ID Nos: 2, 4 or 6, and which encodes a polypeptide which retains at least a portion of the biological activity of the full-length protein as defined herein, or alternatively, which is functional as an antagonist of the biological activity of the full-length protein. For example, such fragments include, as appropriate to the full-length protein from which they are derived, a polypeptide containing a CaLB domain and capable of associating with a phospholipid membrane in a calcium dependent manner, an ATP binding motif, and/or a catalytically active domain, e.g., a hect domain.
Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of such recombinant polypeptides.
As indicated by the examples set out below, a nucleic acid encoding a pub polypeptide may be obtained from mRNA or genomic DNA present in any of a number of mammalian cells in accordance with protocols described herein, as well as those generally known to those skilled in the art. A cDNA encoding a pub polypeptide, for example, can be obtained by isolating total mRNA from a cell, e.g. a mammalian cell, e.g. a human cell. Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. A gene encoding a pub protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
Another aspect of the invention relates to the use of the isolated nucleic acid in xe2x80x9cantisensexe2x80x9d therapy. As used herein, antisense therapy refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding one of the subject pub proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
An antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a pub protein. Alternatively, the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding a pub protein. Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by van der Krol et al., (1988) Biotechniques 6:958-976; and Stein et al., (1988) Cancer Res 48:2659-2668.
Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, the oligomers of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington""s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous for injection, the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank""s solution or Ringer""s solution. In addition, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
In addition to use in therapy, the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind.
In another aspect of the invention, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a subject pub polypeptide and operably linked to at least one regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the polypeptide having an activity of a pub protein. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the pub proteins of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast xcex1-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector""s copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
As will be apparent, the subject gene constructs can be used to cause expression of the subject pub polypeptides in cells propagated in culture, e.g. to produce proteins or polypeptides, including fusion proteins or polypeptides, for purification.
In addition, recombinant expression of the subject pub polypeptides in cultured cells can be useful for controlling differentiation states of cells in vitro, for instance, by controlling the steady state level of activation of cdc25 and thus, the activation of a CDK, e.g., cdc2. To illustrate, in vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors. Once a neuronal cell has become terminally-differentiated, it typically will not change to another terminally differentiated cell-type. However, neuronal cells can nevertheless sometimes lose their differentiated state. This is commonly observed when they are grown in culture from adult tissue, and when they form a blastema during regeneration. By preventing the activation of an M-phase CDK, certain of the pub homologs (presumably agonist forms) can prevent mitotic progression and hence provide a means for ensuring an adequately restrictive environment in order to maintain neuronal cells at various stages of differentiation, and can be employed, for instance, in cell cultures designed to test the specific activities of trophic factors. Other tissue culture systems which require maintenance of differentiation will be readily apparent to those skilled in the art. In this respect, each of the agonist and antagonist of pub activation can be used for ex vivo tissue generation, as for example, to enhance the generation of prosthetic tissue devices for implantation.
To further illustrate, hyper-proliferative cells can be created by antagonizing the activity of the wild-type pub protein, such as by expression of antagonistic homologs, e.g. dominant negative mutants, antisense constructs, or treatment with agents able to disrupt binding of a pub protein with, for example, a cdc25 phosphatase. Pub antagonists provides a method of transforming mammalian cells to be used as in vivo systems to characterize mitotic inhibitors. Conversely, a hypo-proliferative cell can be created by potentiating the activity of the wild type pub protein by expression of agonist homologs or treatment with agents that enhance the binding of pub to cdc25, and thus reduce the level of cdc25 present in a cell.
Moreover, antagonizing the activity of the wild-type pub proteins, such as by expression of antagonistic homologs, antisense constructs, or treatment with agents able to disrupt binding of pub proteins with a cdc25 protein, can be utilized in diagnostic assays to determine if a cell""s growth is no longer dependent on the regulatory function of cdc25 and pub proteins, e.g. in determining the phenotype of a transformed cell. To illustrate, a sample of cells from the tissue can be obtained from a patient and dispersed in appropriate cell culture media, a portion of the cells in the sample can be caused to express a recombinant pub protein, e.g. by transfection with an expression vector, and subsequent growth of the cells assessed. The ability of cells to proliferate despite expression of an agonistic pub protein is indicative of a lack of dependence on cell regulatory pathways which include the pub protein, e.g. a cdc25/cdk-dependent pathway(s). Depending on the nature of the tissue of interest, the sample can be in the form of cells isolated from, for example, a blood sample, an exfoliated cell sample, a fine needle aspirant sample, or a biopsied tissue sample. Where the initial sample is a solid mass, the tissue sample can be minced or otherwise dispersed so that cells can be cultured, as is known in the art. Such knowledge can have both prognostic and therapeutic benefits.
Thus, another aspect of the present invention concerns recombinant pub proteins which have at least one biological activity of a naturally occurring pub protein, or which are naturally occurring mutants thereof. The term xe2x80x9crecombinant proteinxe2x80x9d refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the pub protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. Moreover, the phrase xe2x80x9cderived fromxe2x80x9d, with respect to a recombinant gene encoding the recombinant pub protein, is meant to include within the meaning of xe2x80x9crecombinant proteinxe2x80x9d those proteins having an amino acid sequence of a native pub protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions of a naturally occurring pub protein. To illustrate, recombinant proteins preferred by the present invention, in addition to native pub proteins, are those recombinantly produced proteins which are at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6. Polypeptides having an activity of a pub protein, such as the ability to transfer an ubiquitin molecule form the relevant ubiquitin conjugating enzyme (UBC) or E2 to a lysine residue of its target through a pub ubiquitin thioester intermediate, and having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6 are also within the scope of the invention. Thus, the present invention pertains to recombinant pub proteins which are encoded by genes derived from an eukaryotic cell and which have amino acid sequences evolutionarily related to a pub protein represented by one of SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6, wherein xe2x80x9cevolutionarily related toxe2x80x9d, refers to pub proteins having amino acid sequences which have arisen naturally (e.g. by allelic variance or by differential splicing), as well as mutational variants of pub proteins which are derived, for example, by combinatorial mutagenesis.
This invention also pertains to a host cell transfected with a recombinant pub gene in order to express a polypeptide having an activity of a pub protein. The host cell may be any prokaryotic or eukaryotic cell. For example, a pub protein of the present invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
Accordingly, the present invention further pertains to methods of producing the subject pub proteins. For example, a host cell transfected with an expression vector encoding a pub polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the pub protein. In a preferred embodiment, the pub protein is a fusion protein containing a domain which facilitates its purification, such as a pub-GST fusion protein.
Thus, a nucleotide sequence derived from the cloning of the pub proteins described in the present invention, encoding all or a selected portion of the protein, can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known cell-cycle regulatory proteins, e.g. p53, cyclins, RB, p16, ubc4, E6-AP, and the like. Similar procedures, or modifications thereof, can be employed to prepare recombinant pub proteins, or portions thereof, by microbial means or tissue-culture technology in accord with the subject invention.
The recombinant pub protein can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vehicles for production of a recombinant pub protein include plasmids and other vectors. For instance, suitable vectors for the expression of pub include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. 
A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.
The preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant pub protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the xcex2-gal containing pBlueBac III).
When expression of a carboxy terminal fragment of the full-length pub proteins is desired, i.e. a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well known in the art that a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) Proc. Natl. Acad. Sci. USA 84:2718-1722). Therefore, removal of an N-terminal methionine, if desired, can be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al.).
Alternatively, the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of the pub protein. For example, the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of polypeptide, either in the monomeric form or in the form of a viral particle. The nucleic acid sequences corresponding to the portion of the pub protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the protein as part of the virion. The Hepatitis B surface antigen can also be utilized in this role as well. Similarly, chimeric constructs coding for fusion proteins containing a portion of a pub protein and the poliovirus capsid protein can be created to enhance immunogenicity (see, for example, EP Publication No. 0259149; and Evans et al., (1989) Nature 339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al., (1992) J. Virol. 66:2).
The Multiple Antigen Peptide system for peptide-based immunization can be utilized, wherein a desired portion of a pub protein is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al., (1988) JBC 263:1719 and Nardelli et al., (1992) J. Immunol. 148:914). Antigenic determinants of the pub protein can also be expressed and presented by bacterial cells.
In addition to utilizing fusion proteins to enhance immunogenicity, it is widely appreciated that fusion proteins can also facilitate the expression of proteins. For example, the pub protein of the present invention can be generated as a glutathione-S-transferase (GST) fusion proteins. Such GST fusion proteins can be used to simply purification of the pub protein, such as through the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., (N.Y.: John Wiley and Sons, 1991)).
In another embodiment, a fusion gene coding for a purification leader sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin. The purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified pub protein (e.g., see Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., Proc. Natl. Acad. Sci. USA 88:8972).
Techniques for making fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley and Sons: 1992).
The present invention also makes available isolated and/or purified forms of the subject pub polypeptides, which are isolated from, or otherwise substantially free of other intracellular proteins, especially cell-cycle regulatory proteins, e.g. cdc25 phosphatase or E2 enzymes, which might normally be associated with the pub protein. The term xe2x80x9csubstantially free of other cellular proteinsxe2x80x9d (also referred to herein as xe2x80x9ccontaminating proteinsxe2x80x9d) is defined as encompassing, for example, pub preparations comprising less than 20% (by dry weight) contaminating protein, and preferably comprises less than 5% contaminating protein. Functional forms of the pub polypeptide can be prepared, for the first time, as purified preparations by using a cloned gene as described herein. By xe2x80x9cpurifiedxe2x80x9d, it is meant, when referring to a polypeptide, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins (particularly other cell-cycle proteins such as cdc25 phosphatase, as well as other contaminating proteins). The term xe2x80x9cpurifiedxe2x80x9d as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term xe2x80x9cpurexe2x80x9d as used herein preferably has the same numerical limits as xe2x80x9cpurifiedxe2x80x9d immediately above. xe2x80x9cIsolatedxe2x80x9d and xe2x80x9cpurifiedxe2x80x9d do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.
The subject polypeptides can also be provided in pharmaceutically acceptable carriers for formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington""s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. In an exemplary embodiment, the pub polypeptide is provided for transmucosal or transdermal delivery. For such administration, penetrants appropriate to the barrier to be permeated are used in the formulation with the polypeptide. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
Another aspect of the invention relates to polypeptides derived from the full-length pub protein. Isolated peptidyl portions of the subject pub protein can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such polypeptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, pub protein can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of, for example, cdc25 degradation, such as by microinjection assays. In an illustrative embodiment, peptidyl portions of pub protein can tested for cdc25-binding activity, as well as inhibitory ability, by expression as, for example, thioredoxin fusion proteins, each of which contains a discrete fragment of the pub protein (see, for example, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publication WO94/02502).
It is also possible to modify the structure of the subject pub proteins for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified polypeptides, when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the pub polypeptides described in more detail herein. Such modified polypeptides can be produced, for instance, by amino acid substitution, deletion, or addition.
For instance, it is reasonable to expect, for example, that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine, (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W. H. Freeman and Co., 1981). Whether a change in the amino acid sequence of a polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type protein. For instance, such variant forms of pub can be assessed for their ability to bind to a cdc25 phosphatase of the present invention or other cellular protein. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
This invention further contemplates a method of generating sets of combinatorial mutants of the subject pub proteins, as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that are functional in binding to a regulatory protein, especially cdc25 phosphatase. The purpose of screening such combinatorial libraries is to generate, for example, pub homologs which can act as either agonists or antagonist, or alternatively, which possess novel activities all together. To illustrate, homologs can be engineered by the present method to provide more efficient binding to cdc25 phosphatase, yet have a significantly reduced binding affinity for other cell-cycle regulatory proteins relative to the naturally-occurring form of the protein. Thus, combinatorially-derived homologs can be generated which have a selective potency relative to a naturally occurring pub protein. Such proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols.
Likewise, mutagenesis can give rise to homologs which have intracellular half-lives dramatically different than the corresponding wild-type protein. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of the pub protein. Such homologs, and the genes which encode them, can be utilized to alter the envelope of pub expression by modulating the half-life of the protein. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant pub protein levels within the cell. As above, such proteins, and particularly their recombinant nucleic acid constructs, can be used in gene therapy protocols.
In similar fashion, pub homologs can be generated by the present combinatorial approach to act as antagonists, in that they are able to interfere with the ability of the corresponding wild-type protein to regulate cell proliferation.
In a representative embodiment of this method, the amino acid sequences for a population of pub protein homologs are aligned, preferably to promote the highest homology possible. Such a population of variants can include, for example, homologs from one or more species, or homologs from the same species but which differ due to mutation. Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences. In a preferred embodiment, the combinatorial library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential pub protein sequences. For instance, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential pub nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display).
There are many ways by which the library of potential homologs can be generated from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression. The purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential pub sequences. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) Proc. Natl. Acad. Sci. USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).
Alternatively, other forms of mutagenesis can be utilized to generate a combinatorial library. For example, pub homologs (both agonist and antagonist forms) can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning matagenesis, particularly in a combinatorial setting, is on attractive method for identifying truncated (bioactive) forms of the pub proteins.
A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of pub homologs. The most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate sequences created by combinatorial mutagenesis techniques.
In an illustrative embodiment of a screening assay, candidate pub combinatorial gene products, are displayed on the surface of a cell, and the ability of particular cells or viral particles to bind the cdc25 polypeptide, or other binding partners of pub via this gene product is detected in a xe2x80x9cpanning assayxe2x80x9d. For instance, the pub gene library can be cloned into the gene for a surface membrane protein of a bacterial cell (Ladner et al., WO 88/06630; Fuchs et al., (1991) Bio/Technology 9:1370-1371; and Goward et al., (1992) TIBS 18:136-140), and the resulting fusion protein detected by panning, e.g. using a fluorescently labeled molecule which binds the pub protein, e.g. FITC-cdc25, to score for potentially functional homologs. Cells can be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, separated by a fluorescence-activated cell sorter. While the preceding description is directed to embodiments exploiting the interaction between pub and a cdc25 polypeptide, it will be understood that similar embodiments can be generated using, for example, a pub polypeptide displayed on the surface of a cell and examining the ability of those pub-expressing cells to bind other binding partners of pub.
In similar fashion, the gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at very high concentrations, a large number of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd, and fl are most often used in phage display libraries, as either of the phage gIII or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al., PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al., (1991) Nature 352:624-628; and Barbas et al., (1992) Proc. Natl. Acad. Sci. USA 89:4457-4461).
In an illustrative embodiment, the recombinant phage antibody system (RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified for use in expressing and screening pub combinatorial libraries of the present invention. For instance, the pCANTAB 5 phagemid of the RPAS kit contains the gene which encodes the phage gIII coat protein. The pub combinatorial gene library can be cloned into the phagemid adjacent to the gIII signal sequence such that it will be expressed as a gIII fusion protein. After ligation, the phagemid is used to transform competent E. coli TG1 cells. Transformed cells are subsequently infected with M13KO7 helper phage to rescue the phagemid and its candidate pub gene insert. The resulting recombinant phage contain phagemid DNA encoding a specific candidate pub protein, and display one or more copies of the corresponding fusion coat protein. The phage-displayed candidate proteins which are capable of, for example, binding cdc25, are selected or enriched by panning. For instance, the phage library can be panned on glutathione immobilized cdc25-GST fusion proteins, and unbound phage washed away from the cells. The bound phage is then isolated, and if the recombinant phage express at least one copy of the wild type gIII coat protein, they will retain their ability to infect E. coli. Thus, successive rounds of reinfection of E. coli, and panning will greatly enrich for pub homologs which can then be screened for further biological activities in order to differentiate agonists and antagonists.
Consequently, the invention also provides for reduction of the subject pub proteins to generate mimetics, e.g. peptide or non-peptide agents, which are able to mimic binding of the authentic protein to another cellular partner. Such mutagenic techniques as described above, as well as the thioredoxin system, are also particularly useful for mapping the determinants of a pub protein which participate in protein-protein interactions involved in, for example, binding of the subject proteins to each other. To illustrate, the critical residues of a pub protein which are involved in molecular recognition of cdc25 can be determined and used to generate pub-derived peptidomimetics which bind to cdc25, and by inhibiting pub binding, act to prevent activation of the kinase. By employing, for example, scanning mutagenesis to map the amino acid residues of pub which are involved in binding cdc25, peptidomimetic compounds can be generated which mimic those residues in binding to the kinase. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem. 29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), xcex2-turn dipeptide cores (Nagai et al., (1985) Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans 1:1231), and xcex2-aminoalcohols (Gordon et al., (1985) Biochem Biophys Res Commun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun 134:71).
Another aspect of the invention pertains to an antibody specifically reactive with a pub protein. For example, by using peptides based on the sequence of the subject human or yeast pub protein, anti-pub1 or anit-pub2 antisera or anti-pub1 or anti-pub2 monoclonal antibodies can be made using standard methods. A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. For instance, a peptidyl portion of the protein represented by SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No. 6 can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
Following immunization, anti-pub antisera can be obtained and, if desired, polyclonal anti-pub antibodies isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, an include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497). as the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the pub proteins and the monoclonal antibodies isolated.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with an eukaryotic, e.g., mammalian pub protein. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(abxe2x80x2)2 fragments can be generated by treating antibody with pepsin. The resulting F(abxe2x80x2)2 fragment can be treated to reduce disulfide bridges to produce Fabxe2x80x2 fragments. The antibody of the present invention is further intended to include bispecific and chimeric molecules, as well as single chain (scFv) antibodies.
Both monoclonal and polyclonal antibodies (Ab) directed against the subject pub protein, and antibody fragments such as Fabxe2x80x2 and F(abxe2x80x2)2, can be used to selectively block the action of individual pub proteins and allow the study of the cell-cycle or cell proliferation.
Another application of anti-pub antibodies is in the immunological screening of cDNA libraries constructed in expression vectors, such as xcexgt11, xcexgt18-23, xcexZAP, and xcexORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, xcexgt11 will produce fusion proteins whose amino termini consist of xcex2-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a pub protein, such as proteins antigenically related to the h-pub1 protein of SEQ ID No. 2 or s-pub1 of SEQ ID No. 4 or the h-pub2 protein of SEQ ID No. 6, can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with an anti-pub antibody. Phage, scored by this assay, can then be isolated from the infected plate. Thus, pub homologs can be detected and cloned from other sources.
Antibodies which are specifically immunoreactive with a pub protein of the present invention can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of the protein. Anti-pub antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate levels of one or more pub proteins in tissue or cells isolated from a bodily fluid as part of a clinical testing procedure. Diagnostic assays using anti-pub antibodies, can include, for example, immunoassays designed to aid in early diagnosis of a neoplastic or hyperplastic disorder, e.g. the presence of cancerous cells in the sample, e.g. to detect cells in which alterations in expression levels of pub gene has occurred relative to normal cells.
In addition, nucleotide probes can be generated from the cloned sequence of the subject pub proteins which allow for histological screening of intact tissue and tissue samples for the presence of a pub protein encoding nucleic acids. Similar to the diagnostic uses of anti-pub protein antibodies, the use of probes directed to pub protein encoding mRNAs, or to genomic pub gene sequences, can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, neoplastic or hyperplastic disorders (e.g. unwanted cell growth) or unwanted differentiation events.
Used in conjunction with anti-pub protein antibody immunoassays, the nucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of a pub protein. For instance, variation in pub protein synthesis can be differentiated from a mutation in the coding sequence.
Accordingly, the present method provides a method for determining if a subject is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In preferred embodiments, method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding a pub protein, such as h-pub1 or h-pub2; or (ii) the mis-expression of the pub gene. To illustrate, such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from a pub gene, (ii) an addition of one or more nucleotides to a pub gene, (iii) a substitution of one or more nucleotides of a pub gene, (iv) a gross chromosomal rearrangement of a pub gene, (v) a gross alteration in the level of a messenger RNA transcript of a pub gene, (vii) aberrant modification of a pub gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a pub gene, (viii) a non-wild type level of a pub protein, and (ix) inappropriate post-translational modification of a pub protein. As set out below, the present invention provides a large number of assay techniques for detecting lesions in a pub gene, and importantly, provides the ability to discern between different molecular causes underlying pub dependent aberrant cell growth, proliferation and/or differentiation.
Diagnostic assays are also similarly available for detecting s-pub1 genes, or homologs from other fungus, in order to detect mycotic infections.
In an exemplary embodiment, there is provided a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of a pub gene, such as represented by any of SEQ ID Nos: 1, 3 or 5, or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences or intronic sequences naturally associated with the subject pub genes or naturally occurring mutants thereof. The nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected. Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
In certain embodiments, detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., (1988) Science 241:1077-1080; and Nakazawa et al., (1944) Proc. Natl. Acad. Sci. USA 91:360-364), the later of which can be particularly useful for detecting point mutations in the pub gene. In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to a pub gene under conditions such that hybridization and amplification of the pub gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
In still another embodiment, the level of a pub protein can be detected by immunoassay. For instance, the cells of a biopsy sample can be lysed, and the level of a pub protein present in the cell can be quantitated by standard immunoassay techniques. In yet another exemplary embodiment, aberrant methylation patterns of a pub gene can be detected by digesting genomic DNA from a patient sample with one or more restriction endonucleases that are sensitive to methylation and for which recognition sites exist in the pub gene (including in the flanking and intronic sequences). See, for example, Buiting et al., (1994) Human Mol Genet 3:893-895. Digested DNA is separated by gel electrophoresis, and hybridized with probes derived from, for example, genomic or cDNA sequences. The methylation status of the pub gene can be determined by comparison of the restriction pattern generated from the sample DNA with that for a standard of known methylation.
Furthermore, the subject gene constructs described above can be utilized in diagnostic assays to determine if a cell""s growth is no longer dependent on the regulatory function of a pub protein, e.g. in determining the phenotype of a transformed cell. To illustrate, a sample of cells from the tissue can be obtained from a patient and dispersed in appropriate cell culture media, a portion of the cells in the sample can be caused to express a recombinant pub protein, e.g. by transfection with an h-pub1, h-pub2 or s-pub1 expression vector, and subsequent growth of the cells assessed. The ability of cells to proliferate despite expression of the pub protein is indicative of a lack of dependence on cell regulatory pathways which include the pub protein. Depending on the nature of the tissue of interest, the sample can be in the form of cells isolated from, for example, a blood sample, an exfoliated cell sample, a fine needle aspirant sample, or a biopsied tissue sample. Where the initial sample is a solid mass, the tissue sample can be minced or otherwise dispersed so that cells can be cultured, as is known in the art. Such knowledge can have both prognostic and therapeutic benefits.
In yet another embodiment, a diagnostic assay is provided which detects the ability of a pub gene product, e.g., isolated from a biopsied cell, to bind to other cellular proteins. For instance, it will be desirable to detect h-pub1 mutants which bind with higher binding affinity a cdc25 phosphatase. Such mutants may arise, for example, from fine mutations, e.g., point mutants, which may be impractical to detect by the diagnostic DNA sequencing techniques or by the immunoassays described above. The present invention accordingly further contemplates diagnostic screening assays which generally comprise cloning one or more pub genes from the sample cells, and expressing the cloned genes under conditions which permit detection of an interaction between that recombinant gene product and a target protein, e.g., a cdc25.
As will be apparent from the description of the various drug screening assays set forth below, a wide variety of techniques can be used to determine the ability of a pub protein to bind to other cellular components, e.g., a cdc25 phosphatase such as cdc25A, cdc25B or cdc25C. These techniques can be used to detect mutations in a pub gene which give rise to mutant proteins with a higher or lower binding affinity for a cdc25 relative to the wild-type pub gene product. Conversely, by switching which of the cdc25 and pub protein is the xe2x80x9cbaitxe2x80x9d and which is derived from the patient sample, the subject assay can also be used to detect cdc25 mutants which have a higher or lower binding affinity for a pub protein relative to a wild-type form of that cdc25.
In an exemplary embodiment, cdc25 (e.g. wild-type) can be provided as an immobilized protein (a xe2x80x9cbaitxe2x80x9d or xe2x80x9ctargetxe2x80x9d), such as by use of GST fusion proteins and glutathione-treated microtitre plates. A pub gene (a xe2x80x9csamplexe2x80x9d gene) is amplified from cells of a patient sample, e.g., by PCR, cloned into an expression vector, and transformed into an appropriate host cell. The recombinantly produced pub protein is then contacted with the immobilized cdc25, e.g., as a lysate or a semi-purified preparation (see infra), the complex washed, and the amount of cdc25/pub complex determined and compared to a level of wild-type complex formed in a control. Detection can be by, for instance, an immunoassay using antibodies against the wild-type form of the pub protein, or by virtue of a label provided by cloning the sample pub gene into a vector which provides the protein as a fusion protein including a detectable tag. For example, a myc epitope can provided as part of a fusion protein with the sample pub gene. Such fusion proteins can, in addition to providing a detectable label, also permit purification of the sample pub protein from the lysate prior to application to the immobilized.
In yet another embodiment of the subject screening assay, the two hybrid assay can be used to detect mutations in either a pub gene or cdc25 gene which alter complex formation between those two proteins (see, for example, U.S. Pat. No. 5,283,317; PCT publication WO94/10300; Zervos et al., (1993) Cell 72:223-232; Madura et al., (1993) J Biol Chem 268:12046-12054; Bartel et al., (1993) Biotechniques 14:920-924; and Iwabuchi et al., (1993) Oncogene 8:1693-1696). Accordingly, the present invention provides a convenient method for detecting mutants of pub genes encoding proteins which are unable to physically interact with a cdc25 xe2x80x9cbaitxe2x80x9d protein, which method relies on detecting the reconstitution of a transcriptional activator in a pub/cdc25-dependent fashion.
Still another aspect of the invention features transgenic non-human animals which express a heterologous pub gene of the present invention, or which have had one or more genomic pub gene(s) disrupted in at least one of the tissue or cell-types of the animal. For instance, transgenic mice that are disrupted at their pub gene locus can be generated.
In another aspect, the invention features an animal model for developmental diseases, which has a pub allele which is mis-expressed. For example, a mouse can be bred which has a pub allele deleted, or in which all or part of one or more pub exons are deleted. Such a mouse model can then be used to study disorders arising from mis-expression of the pub gene.
Accordingly, the present invention concerns transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express an exogenous pub protein in one or more cells in the animal. The pub transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs. In preferred embodiments, the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern. In the present invention, such mosaic expression of the subject protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, modulation of cdc25 protein levels, and thus activation of a CDK, e.g., cdc2 which might grossly alter development in small patches of tissue within an otherwise normal embryo. Toward this and, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
Genetic techniques which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are known to those skilled in the art. For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase xe2x80x9ctarget sequencexe2x80x9d refers to a nucleotide sequence that is genetically recombined by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of the subject pub polypeptides. For example, excision of a target sequence which interferes with the expression of a recombinant pub gene can be designed to activate expression of that gene. This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the pub gene from the promoter element or an internal stop codon. Moreover, the transgene can be made wherein the coding sequence of the gene is flanked recombinase recognition sequences and is initially transfected into cells in a 3xe2x80x2 to 5xe2x80x2 orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5xe2x80x2 end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
In an illustrative embodiment, either the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236; Orban et al., (1992) Proc. Natl. Acad. Sci. USA 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O""Gorman et al., (1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination. The orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al., (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
Accordingly, genetic recombination of the target sequence is dependent on expression of the Cre recombinase. Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus, the activation expression of the pub gene can be regulated via regulation of recombinase expression.
Use of the cre/loxP recombinase system to regulate expression of a recombinant pub protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and the recombinant pub genes can be provided through the construction of xe2x80x9cdoublexe2x80x9d transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., the pub gene and recombinase gene.
One advantage derived from initially constructing transgenic animals containing a pub transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein may be deleterious upon expression in the transgenic animal. In such an instance, a founder population, in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues. Thus, the creation of a founder population in which, for example, an antagonistic pub transgene is silent will allow the study of progeny from that founder in which disruption of cell-cycle regulation in a particular tissue or at developmental stages would result in, for example, a lethal phenotype.
Similar conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene. Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Pat. No. 4,833,080. Moreover, expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the pub transgene could remain silent into adulthood until xe2x80x9cturned onxe2x80x9d by the introduction of the trans-activator.
In an exemplary embodiment, the xe2x80x9ctransgenic non-human animalsxe2x80x9d of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pl of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al., (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci. USA 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten et al., (1985) Proc. Natl. Acad. Sci. USA 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al., (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al., (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al., (1982) supra).
A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. USA 83: 9065-9069; and Robertson et al., (1986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:1468-1474.
Methods of making knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of a pub gene can be controlled as above.
Yet another aspect of the invention pertains to methods of treating proliferative and/or differentiative disorders which arise from cells which, despite aberrant growth control, still require a pub-dependent cdc25 activation for cell growth. There are a wide variety of pathological cell proliferative conditions for which the pub gene constructs, pub mimetics and pub antagonists, of the present invention can provide therapeutic benefits, with the general strategy being the inhibition of anomalous cell proliferation. For instance, the gene constructs of the present invention can be used as a part of a gene therapy protocol, such as to reconstitute the function of an h-pub1 or h-pub2 proteins, e.g. in a cell in which the protein is misexpressed or in which signal transduction pathways upstream of a pub protein are dysfunctional, or to inhibit the function of the wild-type protein, e.g. by delivery of a dominant negative mutant.
To illustrate, cell types which exhibit pathological or abnormal growth presumably dependent at least in part on a function (or dysfunction) of a pub protein include various cancers and leukemias, psoriasis, bone diseases, fibroproliferative disorders such as involving connective tissues, atherosclerosis and other smooth muscle proliferative disorders, as well as chronic inflammation. In addition to proliferative disorders, the treatment of differentiative disorders which result from either de-differentiation of tissue due to aberrant reentry into mitosis, or unwanted differentiation due to a failure of a cdc25 phosphatase to appropriately activate certain CDK complexes.
It will also be apparent that, by transient use of gene therapy constructs of the subject pub proteins (e.g. agonist and antagonist forms) or antisense nucleic acids, in vivo reformation of tissue can be accomplished, e.g. in the development and maintenance of organs. By controlling the proliferative and differentiative potential for different cells, the subject gene constructs can be used to reform injured tissue, or to improve grafting and morphology of transplanted tissue. For instance, pub agonists and antagonists can be employed therapeutically to regulate organs after physical, chemical or pathological insult. For example, gene therapy can be utilized in liver repair subsequent to a partial hepatectomy, or to promote regeneration of lung tissue in the treatment of emphysema.
In one aspect of the invention, expression constructs of the subject pub proteins may be administered in any biologically effective carrier. e.g. any formulation or composition capable of effectively transfecting cells in vivo with a recombinant pub gene. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus. and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically.
A preferred approach for in vivo introduction of nucleic acid encoding one of the subject proteins into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed xe2x80x9cpackaging cellsxe2x80x9d) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a pub polypeptide, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include "psgr"Crip, "psgr"Cre. "psgr"2 and "psgr"Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al., (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al., (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
In choosing retroviral vectors as a gene delivery system for the subject pub genes, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the recombinant pub gene, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors to deliver agonistic pub gene constructs. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g. nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.
Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al., (1989) Proc. Natl. Acad. Sci. USA 86:9079-9083; Julan et al., (1992) J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the pub gene of the retroviral vector.
Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al., (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al., (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al., (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127). Expression of the inserted pub gene can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
Yet another viral vector system useful for delivery of the subject pub genes is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993) J. Biol. Chem. 268:3781-3790).
Other viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses. In particular, herpes virus vectors may provide a unique strategy for persistence of the recombinant pub gene in cells of the central nervous system and ocular tissue (Pepose et al., (1994) Invest Ophihalmol Vis Sci 35:2662-2666)
In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a pub protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject pub gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
In a representative embodiment, a gene encoding a pub polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of neuroglioma cells can be carried out using liposomes tagged with monoclonal antibodies against glioma-associated antigen (Mizuno et al., (1992) Neurol. Med. Chir. 32:873-876).
In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as poly-lysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, the subject pub gene construct can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g. poly-lysine (see U.S. Pat. No. 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs viaxe2x80x94mediated endocytosis can be improved using agents which enhance escape of the gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., (1993) Science 260-926; Wagner et al., (1992) Proc. Natl. Acad. Sci. USA 89:7934; and Christiano et al., (1993) Proc. Natl. Acad. Sci. USA 90:2122).
In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the construct in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al., (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057).
Moreover, as set out above, the present invention also provides assays for identifying drugs which are either agonists or antagonists of the normal cellular function of pub proteins, or of the role of pub proteins in the pathogenesis of normal or abnormal cellular proliferation and/or differentiation and disorders related thereto, as mediated by, for example, binding of pub to a target protein, e.g., a mitotic activating tyrosine phosphatase, cdc25. In one embodiment, the assay evaluates the ability of a compound to modulate binding and/or ubiquitinylation of a cdc25 protein or other complexes of cell-cycle regulatory proteins by a pub protein of the present invention. While the following description is directed generally to embodiments exploiting the interaction between pub1 and cdc25, it will be understood that similar embodiments can be generated using, for example, a pub2 protein and cdc25, or either a pub1 or pub2 protein and other cell-cycle regulatory proteins.
A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. Agents to be tested for their ability to act as pub inhibitors can be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. In a preferred embodiment, the test agent is a small organic molecule, e.g., other than a peptide, oligonucleotide, or analog thereof, having a molecular weight of less than about 2,000 daltons.
Assays which approximate the ubiquitination of target regulatory proteins in eukaryotic cells, particularly mammalian cells, can be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Assays as described herein can be used in conjunction with the subject E3-like complexes to generate a ubiquitin-conjugating system for detecting agents able to modulate particular pub-dependent ubiquitination of cellular or viral regulatory proteins. Such modulators can be used, for example, in the treatment of proliferative and/or differentiative disorders, to modulate apoptosis, and in the treatment of viral infections.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as xe2x80x9cprimaryxe2x80x9d screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, potential modifiers, e.g., activators or inhibitors of pub-dependent ubiquitination of a target protein can be detected in a cell-free assay generated by consitution of a functional ubiquitin conjugating system in a cell lysate, such as generated by charging a ubiquitin-depleted reticulocyte lysate (Hershko et al., (1983) J Biol Chem 258:8206-8214) with one or more of a ubiquitin-conjugating enzyme, an E1 enzyme, an E3-like complex comprising pub1, ubiquitin, and/or a substrate for pub1-dependent ubiquitination, such as a cdc25 phosphatase. In an alternate format, the assay can be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.
In an illustrative embodiment of the present assay, the ubiquitin-conjugating system comprises a reconstituted protein mixture of at least semi-purified proteins, and even more preferably of purified proteins. The reconstituted protein mixture is derived from preparations of the regulatory protein and ubiquitin under conditions which drive the conjugation of the two molecules. For instance, the mixture can include a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), an E3-like complex comprising pub1, and a nucleotide triphosphate (e.g. ATP). Alternatively, the E1 enzyme, the ubiquitin, and the nucleotide triphosphate can be substituted in the system with a pre-activated ubiquitin in the form of an E1:Ub conjugate. Likewise, a pre-activated ubiquitin can instead comprise an E2:Ub conjugate.
In preferred embodiments, the purified protein mixture substantially lacks any proteolytic activity which would degrade the target protein and/or components of the ubiquitin conjugating system. For instance, the reconstituted system can be generated to have less than 10% of the proteolytic activity associated with a typical reticulocyte lysate, and preferably no more than 5%, and most preferably less than 2%. Alternatively, the mixture can be generated to include, either from the onset of ubiquitination or from some point after ubiquitin conjugation of the regulatory protein, a ubiquitin-dependent proteolytic activity, such as a purified proteosome complex, that is present in the mixture at measured amounts.
In general, the use of reconstituted protein mixtures will be preferred among cell-free embodiments of the subject assay because they allow more careful control of the reaction conditions in the ubiquitin-conjugating system. Moreover, the system can be derived to favor discovery of modifiers, e.g., activators or inhibitors of particular steps of the ubiquitination process, especially the pub1-dependent steps. For instance, as set out above, a reconstituted protein assay can be generated which does not facilitate degradation of the ubiquitinated protein, and which utilizes a precharged E2:Ub conjugate. The level of ubiquitin-conjugated protein, which is dependent on an E3-like complex can easily be measured directly in such as system, both in the presence and absence of a candidate agent, thereby enhancing the ability to detect a modifier of the pub1-dependent step. Alternatively, the Ub-conjugating system can be allowed to develop a steady state level of regulatory protein:Ub conjugates in the absence of a proteolytic activity, but then shifted to a degradative system by addition of purified Ub-dependent proteases. Such degradative systems would be amenable to identifying proteosome inhibitors.
Moreover, in the subject method, ubiquitin conjugating systems derived from purified proteins hold a number of significant advantages over cell lysate or wheat germ extract based assays (collectively referred to hereinafter as xe2x80x9clysatesxe2x80x9d), especially xe2x80x9cwholexe2x80x9d lysates. Unlike the reconstituted protein system, the synthesis and destruction of the target protein cannot be readily controlled for in lysate-based assays. Without knowledge of particular kinetic parameters for Ub-independant and Ub-dependent degradation of the target protein in the lysate, discerning between the two pathways can be extremely difficult. Measuring these parameters, if at all possible, is further made tedious by the fact that cell lysates tend to be inconsistent from batch to batch, with potentially significant variation between preparations. Evaluation of a potential inhibitor using a lysate system is also complicated in those circumstances where the lysate is charged with mRNA encoding the target protein, as such lysates may continue to synthesize the protein during the assay, and will do so at unpredictable rates.
Accordingly, knowledge of the concentration of each component of the ubiquitin conjugation pathway can be required for each lysate batch, along with the degradative kinetic data, in order to determine the necessary time course and calculate the sensitivity of experiments performed from one lysate preparation to the next.
Furthermore, the lysate system can be unsatisfactory where the target protein itself has a relatively short half-life, especially if due to degradative processes other than the ubiquitin-mediated pathway to which an inhibitor is sought. However, as described, this effect can be mitigated by the use of protease inhibitors such as PMSF or TPCK to inhibit proteolysis of the target protein, though broad-spectrum inhibitors will knock out both ubiquitin-dependent and independent proteolysis.
Moreover, many of the disadvantages of whole cell lysates described above can be overcome by the use of semi-purified cell extracts and/or lysates that have been charged with one or more components of a ubiquitin-conjugation pathway. For example, by selective removal of cell lysate components which interfere with ubiquitination assays, an assay may be feasible in a cell extract even without further purification. Such an approach makes possible rapid and inexpensive development of assay systems suitable for use with ubiquitination assays.
Thus, in another aspect of the subject invention, the ubiquitin-conjugating system comprises a semi-purified cell extract. For instance, as described in the examples below, semi-purified cell extracts can be produced by treatment of cell lysates by a variety of techniques. For example, chromatographic methods and the like can be used to partially purify at least one component of the cell lysate. Likewise, semi-purified cell lysates may be prepared by treatment of a cell lysate to selectively remove a component of the lysate, for example, by immunoprecipitation. Many other methods for the preparation of semi-purified cell extracts by the selective removal or enrichment of components of a cell lysate will be evident to the skilled artisan.
In yet another embodiment of the subject assay, a cell lysate can be charged with certain of the components of a pub1-dependent ubiquitination system. For example, in addition to inhibitors or potentiators of ubiquitination, a semi-purified cell extract can be charged with the relevant UBC, pub1, cdc25 phosphatase and the like. Likewise, lysates can be generated from cells recombinantly manipulated to produce, for example, a labeled component to the assay, such as a myc-labeled ubiquitin or a GST-cdc25 fusion protein.
Ubiquitination of the target regulatory protein via an in vitro ubiquitin-conjugating system, in the presence and absence of a candidate inhibitor, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In certain embodiments of the present assay, the in vitro assay system is generated to lack the ability to degrade the ubiquitinated target protein. In such an embodiments, a wide range of detection means can be practiced to score for the presence of the ubiquitinated protein.
In one embodiment of the present assay, the products of a non-degradative ubiquitin-conjugating system are separated by gel electrophoresis, and the level of ubiquitinated target protein assessed, using standard electrophoresis protocols, e.g., by detecting an increase in molecular weight of the target protein that corresponds to the addition of one or more ubiquitin chains. For example, one or both of the target protein and ubiquitin can be labeled with a radioisotope such as 35S, 14C, or 3H, and the isotopically labeled protein bands quantified by autoradiographic techniques. Standardization of the assay samples can be accomplished, for instance, by adding known quantities of labeled proteins which are not themselves subject to ubiquitination or degradation under the conditions which the assay is performed. Similarly, other means of detecting electrophoretically separated proteins can be employed to quantify the level of ubiquitination of the regulatory protein, including immunoblot analysis using antibodies specific for either the regulatory protein or ubiquitin, or derivatives thereof. As described below, the antibody can be replaced with another molecule able to bind one of either the regulatory protein or ubiquitin. By way of illustration, one embodiment of the present assay comprises the use of biotinylated ubiquitin in the conjugating system. The biotin label is detected in a gel during a subsequent detection step by contacting the electrophoretic products (or a blot thereof) with a streptavidin-conjugated label, such as a streptavidin linked fluorochrome or enzyme, which can be readily detected by conventional techniques. Moreover, where a reconstituted protein mixture is used (rather than a lysate) as the conjugating system, it may be possible to simply detect the regulatory protein and ubiquitin conjugates in the gel by standard staining protocols, including coomassie blue and silver staining.
In another embodiment, an immunoassay or similar binding assay, is used to detect and quantify the level of ubiquitinated regulatory protein produced in the ubiquitin-conjugating system. Many different immunoassay techniques are amenable for such use and can be employed to detect and quantitate the regulatory protein:Ub conjugates. For example, the wells of a microtitre plate (or other suitable solid phase) can be coated with an antibody which specifically binds one of either the regulatory protein or ubiquitin. After incubation of the ubiquitin-conjugated system with and without the candidate agent, the products are contacted with the matrix bound antibody, unbound material removed by washing, and ubiquitin conjugates of the regulatory protein specifically detected. To illustrate, if an antibody which binds the regulatory protein is used to sequester the protein on the matrix, then a detectable anti-ubiquitin antibody can be used to score for the presence of ubiquitinated regulatory protein on the matrix.
However, it will be clear to those skilled in the art that the use of antibodies in these binding assays is merely illustrative of binding molecules in general, and that the antibodies are readily substituted in the assay with any suitable molecule that can specifically detect one of either the regulatory protein or the ubiquitin. As described below, a biotin-derivative of ubiquitin can be used, and streptavidin (or avidin) employed to bind the biotinylated ubiquitin. In an illustrative embodiment, wells of a microtitre plate are coated with streptavidin and contacted with the developed ubiquitin-conjugating system under conditions wherein the biotinylated ubiquitin binds to and is sequestered in the wells. Unbound material is washed from the wells, and the level of regulatory protein (bound to the matrix via a conjugated ubiquitin moiety) is detected in each well. Alternatively, the microtitre plate wells can be coated with an antibody (or other binding molecule) which binds and sequesters the regulatory protein on the solid support, and detection of ubiquitinated conjugates of the matrix-bound regulatory protein are subsequently carried out using a detectable streptavidin derivative, such as an alkaline phosphatase/streptavidin complex.
In similar fashion, epitope-tagged ubiquitin, such as myc-ub (see Ellison et al. (1991) J. Biol. Chem. 266:21150-21157; ubiquitin which includes a 10-residue sequence encoding a protein of c-myc) can be used in conjunction with antibodies to the epitope tag. A major advantage of using such an epitope-tagged ubiquitin approach for detecting Ub:protein conjugates is the ability of an N-terminal tag sequences to inhibit ubiquitin-mediated proteolysis of the conjugated regulatory protein.
Other ubiquitin derivatives include detectable labels which do not interfere greatly with the conjugation of ubiquitin to the regulatory protein. Such detectable labels can include fluorescently-labeled (e.g. FITC) or enzymatically-labeled ubiquitin fusion proteins. These derivatives can be produced by chemical cross-linking, or, where the label is a protein, by generation of a fusion protein. Several labeled ubiquitin derivatives are commercially available.
Likewise, other binding molecules can be employed in place of the antibodies that bind the regulatory protein. For example, the regulatory protein can be generated as a glutathione-S-transferase (GST) fusion protein. As a practical matter, such GST fusion protein can enable easy purification of the regulatory protein in the preparation of components of the ubiquitin-conjugating system (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (NY: John Wiley and Sons, 1991); Smith et al. (1988) Gene 67:31; and Kaelin et al. (1992) Cell 70:351) Moreover, glutathione derivatized matrices (e.g. glutathione-sepharose or glutathione-coated microtitre plates) can be used to sequester free and ubiquitinated forms of the regulatory protein from the ubiguitin-conjugating system, and the level of ubiquitin immobilized can be measured as described. Likewise, where the matrix is generated to bind ubiquitin, the level of sequestered GST-regulatory protein can be detected using agents which bind to the GST moiety (such as anti-GST antibodies), or, alternatively, using agents which are enzymatically acted upon by GST to produce detectable products (e.g. 1-chloro-2,4-dinitrobenzene; Habig et al. (1974) J Biol Chem 249:7130). Similarly, other fusion proteins involving the regulatory protein and an enzymatic activity are contemplated by the present method. For example, fusion proteins containing xcex2-galactosidase or luciferase, to name but a few, can be employed as labels to determine the amount of regulatory protein sequestered on a matrix by virtue of a conjugated ubiquitin chain.
Moreover, such enzymatic fusion proteins can be used to detect and quantitate ubiquitinated regulatory protein in a heterogeneous assay, e.g., one which does not require separation of the components of the conjugating system. For example, ubiquitin conjugating systems can be generated to have a ubiquitin-dependent protease which degrades the regulatory protein. The enzymatic activity of undegraded fusion protein provides a detectable signal, in the presence of substrate, for effectively measuring the level of the regulatory protein ubiquitination. Similarly, in a non-degradative conjugating system, ubiquitination of the regulatory protein portion of the fusion protein can allosterically influence the enzymatic activity associated with the fusion the protein and thereby provides a means for monitoring the level of ubiquitin conjugation.
In binding assay-type detection steps such as set out above, the choice of which of either the regulatory protein or ubiquitin should be specifically sequestered on the matrix will depend on a number of factors, including the relative abundance of both components in the conjugating system. For instance, where the reaction conditions of the ubiquitin conjugating system provide ubiquitin at a concentration far in excess of the level of the regulatory protein, (e.g., one order of magnitude or greater) sequestering the ubiquitin and detecting the amount of regulatory protein bound with the ubiquitin can provide less dynamic range to the detection step of the present method than the converse embodiment of sequestering the regulatory protein and detecting ubiquitin conjugates from the total regulatory protein bound to the matrix. That is, where ubiquitin is provided in great excess relative to the regulatory protein, the percentage of ubiquitin conjugated regulatory protein in the total ubiquitin bound to the matrix can be small enough that any diminishment in ubiquitination caused by a modifier can be made difficult to detect by the fact that, for example, the statistical error of the system (e.g. the noise) can be a significant portion of the measured change in concentration of bound regulatory protein. Furthermore, it is clear that manipulating the reaction conditions and reactant concentrations in the ubiquitin-conjugating system can be carried out to provide, at the detection step, greater sensitivity by ensuring that a strong ubiquitinated protein signal exists in the absence of any modifier.
In still further embodiments of the present invention, the ubiquitin-conjugating system is generated in whole cells, taking advantage of cell culture techniques to support the subject assay. For example, as described below, the ubiquitin-conjugating system (including the target protein and detection means) can be constituted in a eukaryotic cell culture system, including mammalian and yeast cells. Advantages to generating the subject assay in an intact cell include the ability to detect inhibitors which are functional in an environment more closely approximating that which therapeutic use of the inhibitor would require, including the ability of the agent to gain, entry into the cell. Furthermore, certain of the in vivo embodiments of the assay, such as examples given below, are amenable to high through-put analysis of candidate agents.
The components of the ubiquitin-conjugating system, including the regulatory protein, can be endogenous to the cell selected to support the assay. Alternatively, some or all of the components can be derived from exogenous sources. For instance, a recombinantly produced E2 enzyme, such as UBC3, UBC4, UBC5 and/or UBC9, or recombinantly produced components of an E3-like complex comprising pub1, can be expressed in the cell by recombinant techniques (such as through the use of an expression vector), as well as by microinjecting the proteins themselves or mRNA encoding the protein.
In any case, the cell is ultimately manipulated after incubation with a candidate inhibitor in order to facilitate detection of ubiquitination or ubiquitin-mediated degradation of the regulatory protein. As described above for assays performed in reconstituted protein mixtures or lysates, the effectiveness of a candidate inhibitor can be assessed by measuring direct characteristics of the regulatory protein, such as shifts in molecular weight by electrophoretic means or detection in a binding assay. For these embodiments, the cell will typically be lysed at the end of incubation with the candidate agent, and the lysate manipulated in a detection step in much the same manner as might be the reconstituted protein mixture or lysate.
Indirect measurement of ubiquitination of the target protein can also be accomplished by detecting a biological activity associated with the regulatory protein that is either attenuated by ubiquitin-conjugation or destroyed along with the regulatory protein by ubiquitin-dependent proteolytic processes. As set out above, the use of fusion proteins comprising the regulatory protein and an enzymatic activity are representative embodiments of the subject assay in which the detection means relies on indirect measurement of ubiquitination of the regulatory protein by quantitating an associated enzymatic activity.
Where the regulatory protein has a relatively short half-life due to ubiquitin-dependent or independent degradation in the cell, preferred embodiments of the assay either do not require cell lysis, or, alternatively, generate a longer lived detection signal that is independent of the regulatory protein""s fate after lysis of the cell. With respect to the latter embodiment, the detection means can comprise, for example, a reporter gene construct which includes a positive transcriptional regulatory element that binds and is responsive to the regulatory protein. For instance, where the regulatory protein does not itself posses DNA-binding ability, it can be arranged as part of an interaction trap assay designed for detecting modifiers, e.g., activators or inhibitors, of the pub1-dependent destruction of the protein (see, for example, U.S. Pat. No. 5,283,317; PCT publication WO94/10300; Zervos et al., (1993) Cell 72:223-232; Madura et al., (1993) J Biol Chem 268:12046-12054; Bartel et al., (1993) Biotechniques 14:920-924; and Iwabuchi et al., (1993) Oncogene 8:1693-1696). In an illustrative embodiment, Saccharomyces cerevisiae YPB2 cells are transformed simultaneously with a plasmid encoding a GAL4db-pub1 (where pub is a catalytically inactive) fusion and with a plasmid encoding the GAL4ad domain fused to human cdc25 phosphatase. Moreover, the strain is transformed such that the GAL4-responsive promoter drives expression of a phenotypic marker. For example, the ability to grow in the absence of histidine depends on the expression of the HIS3 gene if it is under control of a GAL4-responsive promoter and, therefore, indicates that a functional GAL4 activator has been reconstituted through the interaction of the h-pub1 and the human cdc25 fusion proteins.
Thus, for example, agents able to inhibit the ubiquitination of the cdc25 fusion protein will result in yeast cells able to growth in the absence of histidine, as the GAL4db-pub1 and GAL4ad-cdc25 fusion proteins will be able to interact and cause expression of the HIS3 gene. Alternatively, the agents which do not effect the ubiquitination of the cdc25 fusion protein will result in cells unable to grow in the absence of histidine as the GAL4ad-cdc25 fusion protein will be degraded or otherwise prevented from interacting with the GAL4db-pub1 protein.
The present invention also makes available S. pombe strains which contain a null pub mutation. As described herein, these strains can be complemented using human genes, and thus xe2x80x9chumanizedxe2x80x9d yeast strains can be created for in vivo drug screen, e.g., which comprise a human pub homolog and (optionally) a human cdc25 phosphatase. The strain can be further manipulated to be xe2x80x9chumanizedxe2x80x9d with respect to other biochemical steps in the pub1-mediated ubiquitination of the cdc25 fusion protein. For example, conditional inactivation of the relevant yeast UBC enzyme with concomitant expression of the human UBC homolog, or alternatively, replacement of other yeast genes involved in ubiquitination with their human homologs, provides a humanized system whereby the cdc25 protein can be ubiquitinated by a pub1-dependent mechanism which approximates the pub1-dependent ubiquitination that occurs in vertebrate cells.
Furthermore, drug screening assays can be generated which do not measure ubiquitination per se, but rather detect inhibitory agents on the basis of their ability to interfere with binding of one of the proteins involved in the pub1-dependent ubiquitin conjugation pathway. In an exemplary binding assay, the compound of interest is contacted with a mixture generated from an isolated and purified E2 protein and an E3-like complex comprising the pub protein. Alternatively, pub and cdc25 are combined in the presence and absence of test agents so as to provide a competitive binding assay which detects agents able to compete with, or potentiate, the cdc25 binding to pub1. Detection and quantification of complexes between the pub and cdc25 provides a means for determining the compound""s efficacy at inhibiting (or potentiating) complex formation between the pub and other components of the pub1-dependent ubiquitin pathway. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified cdc25 is added to a composition containing the pub protein, and the formation of complexes is quantitated in the absence of the test compound.
Complex formation between cdc25 protein or other regulatory protein and pub may be detected by a variety of techniques, many of which are effectively described above. For instance, modulation in the formation of complexes can be quantitated using, for example, detectably labeled proteins (e.g. radiolabelled, fluorescently labelled, or enzymatically labelled), by immunoassay, or by chromatographic detection.
Typically, it will be desirable to immobilize either the regulatory protein, e.g., cdc25 or a component of the E3-like complex, such as the pub protein, to facilitate separation of complexes from uncomplexed forms of one of the proteins, as well as to accommodate automation of the assay. In an illustrative embodiment, a fusion protein can be provided which adds a domain that permits the protein to be bound to an insoluble matrix. For example, GST/cdc25 fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the pub protein, e.g. containing 35S-labeled proteins, and the test compound and incubated under conditions conducive to complex formation. Following incubation, the beads are washed to remove any unbound pub1, and the matrix bead-bound radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are dissociated, e.g. when microtitre plaste is used. Alternatively, after washing away unbound protein, the complexes can be dissociated from the matrix, separated by SDS-PAGE gel, and the level of, for example, pub protein found in the matrix-bound fraction quantitated from the gel using standard electrophoretic techniques.